US20060115806A1 - Global analysis of transposable elements as molecular markers of cancer - Google Patents

Global analysis of transposable elements as molecular markers of cancer Download PDF

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US20060115806A1
US20060115806A1 US10/554,711 US55471104A US2006115806A1 US 20060115806 A1 US20060115806 A1 US 20060115806A1 US 55471104 A US55471104 A US 55471104A US 2006115806 A1 US2006115806 A1 US 2006115806A1
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John McDonald
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OVARIAN CANCER INSTITUTE
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates to the determination of expression patterns, DNA methylation patterns and chromatin properties of families of transposable elements in order to detect, classify, characterize and treat cancer.
  • the human genome comprises numerous families of transposable elements, such as DNA elements, i.e. Charlie- and Tigger groups (see Smit (1999) Interspersed repeats and other mementos of transposable elements in mammalian genomes. Current Opinion in Genetics & Development, 9: 657-663) and retroelements, i.e., LINEs (long interspersed nuclear elements), SINES (short interspersed nuclear elements) and HERVs (human endogenous retroviruses). To date, over 50 families of retroviral elements have been identified and the members of these families make up greater than 43% of the genome (See Li et al. (2001) Evolutionary analysis of the human genome. Nature, 409 (6822): 847-9).
  • retroelements Some families can include hundreds to thousands of retroelements and the expression of retroelements genes is normally suppressed. However, under certain conditions, such as cancer, retroelements may no longer be suppressed and expression of retroelement genes is activated, concomitant with changes in DNA methylation patterns and/or chromatin states.
  • the present invention provides methods of determining patterns of transposable element expression, transposable element methylation and chromatin status of transposable elements within the genome such that these patterns can be used to diagnose cancer, identify a type of cancer, classify a cancer at a particular stage and measure progression of cancer. All of the methods of the present invention can be utilized to analyze full-length transposable element sequences or fragments thereof. These transposable elements include retroelements and fragments thereof as well as DNA elements and fragments thereof from mammalian species.
  • the present invention provides methods of determining patterns of retroelement expression, retroelement methylation and chromatin status of retroelements within the genome such that these patterns can be used to diagnose cancer, identify a type of cancer, classify a cancer at a particular stage and measure progression of cancer. Also provided are methods of determining DNA element expression, DNA element methylation and chromatin state of DNA elements within the genome such that these patterns can be used to diagnose cancer, identify a type of cancer, classify a cancer at a particular stage and measure progression of cancer.
  • the present invention provides a method of determining an expression pattern of one or more families of transposable elements in a sample comprising determining expression of one or more families of transposable elements.
  • Also provided by the present invention is a method of assigning an expression pattern of transposable elements to a type of cancerous cell in a sample, comprising: a) determining expression of one or more families of transposable elements; and b) assigning the expression pattern obtained from step a) to the type of cancerous cell in the sample.
  • a method of diagnosing cancer comprising: a) determining expression of one or more families of transposable elements in a sample to obtain an expression pattern; b) matching the expression pattern of step a) with a known expression pattern for a type of cancer; and c) diagnosing the type of cancer based on matching of the expression pattern of a) with a known expression pattern for a type of cancer.
  • the present invention also provides a method of determining the effectiveness of an anti-cancer therapeutic in a subject comprising: a) determining expression of one or more families of transposable elements, in a sample obtained from the subject, to obtain a first expression pattern; b) administering an anti-cancer therapeutic to the subject; c) determining expression of one or more families of transposable elements in a sample obtained from the subject after administration of an anti-cancer therapeutic to obtain a second expression pattern; and d) comparing the second expression pattern with the first expression pattern such that if transposable elements are differentially expressed in the second expression pattern as compared to the first expression pattern, the anti-cancer therapeutic is an effective anti-cancer therapeutic.
  • Also provided by the present invention is a method of determining a methylation pattern of one or more families of transposable elements in a sample comprising determining methylation of one or more families of transposable elements.
  • the present invention also provides a method of assigning a methylation pattern of transposable elements to a type of cancerous cell in a sample, comprising: a) determining methylation of one or more families of transposable elements; and b) assigning the methylation pattern obtained from step a) to the type of cancerous cell in the sample.
  • Also provided by the present invention is a method of diagnosing cancer comprising: a) determining methylation of one or more families of transposable elements in a sample to obtain a methylation pattern; b) comparing the methylation pattern of step a) with a known methylation pattern for a type of cancer; and c) diagnosing the type of cancer based on matching of the methylation pattern of a) with a known methylation pattern for a type of cancer.
  • the present invention also provides a method of determining the effectiveness of an anti-cancer therapeutic in a subject comprising: a) determining methylation of one or more families of transposable elements, in a sample obtained from the subject, to obtain a first methylation pattern; b) administering an anti-cancer therapeutic to the subject; c) determining methylation of one or more families of transposable elements in a sample obtained from the subject after administration of an anti-cancer therapeutic to obtain a second methylation pattern; and d) comparing the second methylation pattern with the first methylation pattern such that if there is a change in the second methylation pattern as compared to the first methylation pattern, the anti-cancer therapeutic is an effective anti-cancer therapeutic.
  • FIG. 1 shows RT-PCR from normal and tumor ovarian samples comparing expression levels of HERV-K and HERV-W.
  • ( ⁇ ) indicates a control without reverse transcriptase documenting absence of relevant DNA contamination. No Herv K or Herv W expression was detectable in this normal sample, HervW expression and even higher HervK expression was detected in this ovarian carcinoma sample.
  • FIG. 2 is a southern blot analysis of genomic DNA after digest with MspI (N) or its methylation-sensitive isoschizomer HpaII (H), resp., hybridized with a HERV-W probe spanning the putative promoter region of the element. Equal amounts of DNA were loaded per sample, i.e. MspI/HpaII pair. Fragment sizes range from >0.1 kb to >3.0 kb. Samples represent ovarian carcinoma (T—malignant), ovarian adenoma (B—benign), borderline ovarian tumor (LMP) and non-tumor ovarian tissue (N).
  • T malignant
  • B borderline ovarian tumor
  • N non-tumor ovarian tissue
  • Fragments between 0.3 kb and 1 kb appear in most of the malignant samples in the HpaII digests, but not in adenoma, borderline or non-tumor samples, indicating extensive cytosine methylation of this particular HervW region in non-carcinoma ovarian tissue and loss of HervW methylation in ovarian carcinoma. See region defined by arrows.
  • FIG. 3 is a southern blot analysis of genomic DNA after digest with MspI (M) or its methylation-sensitive isoschizomer HpaII (H), resp., hybridized with a LINE1 probe spanning the putative promoter region of the element. Equal amounts of DNA were loaded per sample, i.e. per MspI/HpaII pair. Fragment sizes range from 0.1 kb to >3.0 kb. Samples represent ovarian carcinoma (T—malignant), borderline ovarian tumor (B) and non-tumor ovarian tissue (N).
  • T malignant
  • B borderline ovarian tumor
  • N non-tumor ovarian tissue
  • FIG. 4 shows hypomethylation and expression of L1 and HERV-W elements in ovarian cancer.
  • Genomic DNA was digested either with MspI (left) or HpaII (right), and hybridized with probes specific for the promoter regions of L1 (A) or HERV-W (B) elements.
  • the restriction enzymes MspI and HpaII recognize the sequence CCGG but HpaII only cuts when the recognition sequence is unmethylated at the inner cytosine (i.e., C C GG) while MspI is indifferent to the methylation status of the inner cytosine.
  • C Real time RT-PCR was performed to determine expression levels of LINE-1 and HERV-W elements in representative malignant and non-malignant samples. Normalized values (retroelement expression value divided by expression value of the RPS27A control gene. Shown is the average of 3 replicate assays per sample USE. Ribosomal protein S27A (RPS27A) expression has been previously determined to be unchanged between the malignant and non-malignant samples examined in this study.
  • FIG. 5 is an example of an array that was utilized to assess retroelements patterns in cancer cells.
  • Each dot represents a hybridization of the labeled RNA pool (from either a cancer or control sample in this case a cancer sample) to the “spots” representing retroelement sequences. A bright color indicates that the element was expressed in this sample. The intensity of the dot is correlated with the level of expression.
  • 3 replicate copies of the elements (spots) are aligned vertically. Different elements families are arranged side by side.
  • nucleic acid includes multiple copies of the nucleic acid and can also include more than one particular species of nucleic acid molecule.
  • a cell includes one or more cells, including populations of cells.
  • the present invention provides a method of determining an expression pattern of one or more families of transposable elements in a sample comprising determining expression of one or more families of transposable elements.
  • sample can be from any organism and can be, but is not limited to, peripheral blood, plasma, urine, saliva, gastric secretion, feces, bone marrow specimens, primary tumors, metastatic tissue, embedded tissue sections, frozen tissue sections, cell preparations, cytological preparations, exfoliate samples (e.g., sputum), fine needle aspirations, amino cells, fresh tissue, dry tissue, and cultured cells or tissue.
  • biological sample of this invention can also be whole cells or cell organelles (e.g., nuclei).
  • the sample can be unfixed or fixed according to standard protocols widely available in the art and can also be embedded in a suitable medium for preparation of the sample.
  • the sample can be embedded in paraffin or other suitable medium (e.g., epoxy or acrylamide) to facilitate preparation of the biological specimen for the detection methods of this invention.
  • the sample can be from a subject or a patient.
  • the “subject” or “patient” of the methods described herein can be any animal.
  • the animal of the present invention is a human.
  • determination of expression patterns is also contemplated for non-human animals which can include, but are not limited to, cats, dogs, birds, horses, cows, goats, sheep, guinea pigs, hamsters, gerbils, mice and rabbits.
  • the sample can comprise a cell or cells selected from the group consisting of: a carcinoma cell, a fibroma cell, a sarcoma cell, a teratoma cell, a blastoma cell, a breast tumor cell of epithelial origin, an ovarian tumor cell of epithelial, stromal or germ cell origin, mixed cell types from a tumor or any other cancer cell.
  • the present invention also provides for the analysis of a sample comprising a normal cell or normal cells from a particular tissue. The patterns obtained from normal cells can be compared to the expression patterns for cancerous cells in order to access the differences between normal and cancerous cells.
  • cancer when used herein refers to or describes the physiological condition, preferably in a mammalian subject, that is typically characterized by unregulated cell growth.
  • examples of cancer include but are not limited to ras-induced cancers, colorectal cancer, carcinoma, lymphoma, sarcoma, blastoma and leukemia. More particular examples of such cancers include squamous cell carcinoma, lung cancer, pancreatic cancer, cervical cancer, bladder cancer, hepatoma, breast cancer, prostrate carcinoma, rhabdomyosarcoma, colon carcinoma, ovarian cancer and head and neck cancer.
  • cancer as used herein is not limited to any one specific form of the disease, it is believed that the methods of the invention will be particularly effective for cancers which are found to be accompanied by changes in transposable element expression, transposable element methylation and/or changes in chromatin status of transposable elements.
  • transposable element families that can be analyzed by the methods of the present invention, including, but not limited to, retroelement families and DNA element families.
  • retroelement families that can be analyzed utilizing the methods of this invention include but are not limited to, endogenous retroviruses (ERVs), short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs), the vertebrate long terminal repeat (LTR)-containing elements, and the poly(A) retrotransposons.
  • the DNA element families that can be analyzed by the methods of the present invention include, but are not limited to the Mariner/Tci superfamily (e.g.
  • retroelement families human Mariner, Tigger, Marna, Golem, Zombi), hAT (hobo/Activator/Tam3) superfamily, TTAA superfamily (e.g. Looper), MITEs (e.g. MER85), MuDR superfamily (e.g. Ricksha), T2-family (E.G. Kanga 2) and others. Any combination of retroelement families and the members of these retroelement families can be analyzed by the methods of the present invention to determine a pattern of expression, a retroelement methylation pattern and/or a retroelement chromatin status pattern.
  • any combination of families and members of transposable element families may be analyzed to provide an expression pattern, chromatin status pattern and/or a methylation pattern. Therefore, combinations of retroelement families and DNA element families can also be also analyzed by the methods of the present invention.
  • a publicly available database, RepBase Update contains consensus sequences of genomic repeats from different organisms that can be utilized to design the oligonucleotides utilized in the methods of the present invention. This database can be accessed at www.girinst.org. This database was utilized to identify consensus sequences for numerous retroelements which were then used to design oligonucleotide probes for the microarrays of the present invention.
  • RepBase Update containing human-specific repeats (consensus sequences for transposon families). Selected RepBase files were then input into the OligoArray program, a publicly available software tool for microarray oligo-design at http://berry.engin.umich.edu/oligoarray and the design algorithm was run.
  • the BLAST algorithm at http://www.ncbi.nlm.nih.gov/BLAST/ (Altschul S F, Gish W, Miller W, Myers E W, Lipman D J Basic local alignment search tool . in J Mol Biol Oct.
  • microarray can be a chip, a glass slide or a nylon membrane comprising different types of material, such as, but not limited to, nucleic acids, proteins or tissue sections.
  • a plurality of transposable element sequences from transposable element families can be analyzed simultaneously to obtain expression patterns.
  • One of skill in the art can design a microarray chip or glass slide that contains the representative nucleic acid sequences of all of the members of a particular transposable element family or the nucleic acid sequences of select members of a particular transposable element family.
  • An array can also contain the nucleic acid sequences of selected transposable elements from one or more families.
  • Array design will vary depending on the transposable element families and the sequences from these families being analyzed.
  • One of skill in the art will know how to design or select an array that contains the transposable element sequences associated with a particular type of cancer.
  • Such microarrays can be obtained from commercial sources such as Affymetrix, or the microarrays can be synthesized. Methods for synthesizing such arrays containing nucleic acid sequences are known in the art. See, for example, U.S. Pat. No. 6,423,552, U.S. Pat. No. 6,355,432 and U.S. Pat. No. 6,420,169 which are hereby incorporated in their entireties by this reference.
  • the present invention also provides microarray slides or chips comprising transposable element sequences or fragments thereof from transposable element families.
  • a microarray slide or chip can contain the representative nucleic acid sequences of all of the members of one or more transposable element families or the nucleic acid sequences of select members of one or more transposable element families.
  • the present invention also provides for a kit comprising a microarray slide or chip of the present invention for diagnosis of cancer, staging of cancer, other clinical applications and research applications. Utilizing the methods of the present invention, a chip(s) or glass slide(s) that specifically detect a type of cancer can be synthesized.
  • transposable element sequences from two families are expressed in prostate cancer
  • a chip that contains the necessary transposable element sequences from these two families can be synthesized, such that one of skill in the art can utilize a kit, containing this chip, for detecting and staging prostate cancer.
  • utilizing the expression patterns of transposable element sequences for breast cancer it is possible to manufacture a kit containing a chip comprising the transposable element sequences involved in breast cancer in order to diagnose and stage breast cancer.
  • utilizing the expression patterns of transposable element sequences for ovarian cancer it is possible to manufacture a kit containing a chip comprising the transposable element sequences involved in ovarian cancer in order to diagnose and stage ovarian cancer.
  • Microarray techniques would be known to one of skill in the art.
  • U.S. Pat. No. 6,410,229 and U.S. Pat. No. 6,344,316 both hereby incorporated by this reference, describe methods of monitoring expression by hybridization to high density nucleic acid arrays.
  • one skilled in the art would first produce fluorescent-labeled cDNAs from mRNAs isolated from cancer cells.
  • a mixture of the labeled cDNAs from the cancer cells is added to an array of oligonucleotides representing a plurality of known transposable elements, as described above, under conditions that result in hybridization of the cDNA to complementary-sequence oligonucleotides in the array.
  • the array is then examined by fluorescence under fluorescence excitation conditions in which transposable element polynucleotides in the array that are hybridized to cDNAs derived from the cancer cells can be detected and quantified.
  • the expression patterns of the present invention can also be determined by assaying for mRNA transcribed from transposable elements, assaying for proteins expressed from a mRNA, RT-PCR and northern blotting. Particular protein products translated from mRNAs transcribed by transposable element genes can be detected by utilizing immunohistochemical techniques, ELISA, 2-D gels, mass spectrometry, Western blotting, and enzyme assays.
  • patterns of expression can include one, two, three, four, five, six, seven, eight, nine, ten, twenty or more families of transposable elements and at least one, two, three, four, five, ten, fifteen, twenty, twenty-five, fifty, one hundred, two hundred, three hundred, four hundred, five hundred, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members of each transposable element family are being analyzed.
  • the present invention provides for the determination of an expression pattern of one family of transposable elements in which one, two, three, four, five, ten, fifteen, twenty, twenty five, fifty, one hundred, two hundred, three hundred, four hundred, five hundred, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members of a transposable element family are analyzed.
  • the present invention also provides for the determination of an expression pattern of two families, wherein one, two, three, four, five, ten, fifteen, twenty, twenty five fifty, one hundred, two hundred, three hundred, four hundred, five hundred, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members are analyzed for each family.
  • the invention provides for the determination of an expression pattern of three families, wherein one, two, three, four, five, ten, fifteen, twenty, twenty five fifty, one hundred, two hundred, three hundred, four hundred, five hundred, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members are analyzed for each family.
  • the invention provides for the determination of an expression pattern of multiple families, for example, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 families wherein one, two, three, four, five, ten, fifteen, twenty, twenty five fifty, one hundred, two hundred, three hundred, four hundred, five hundred, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members are analyzed for each family.
  • families for example, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 families wherein one, two, three, four, five, ten, fifteen, twenty, twenty five fifty, one hundred, two hundred, three hundred, four hundred, five hundred, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven
  • the present invention provides a method of assigning an expression pattern of transposable elements to a type of cancerous cell in a sample, comprising: a) determining expression of one or more families of transposable elements; and assigning the expression pattern obtained from step a) to the type of cancerous cell in the sample.
  • the present invention also provides a method of diagnosing cancer comprising: a) determining expression of one or more families of transposable elements in a sample to obtain an expression pattern; b) matching the expression pattern of step a) with a known expression pattern for a type of cancer, and c) diagnosing the type of cancer based on matching of the expression pattern of a) with a known expression pattern for a type of cancer.
  • the expression pattern obtained from a sample taken from a subject can be obtained from outside sources, such as a testing laboratory or a commercial source. Therefore, the step of obtaining the expression pattern can be performed by one skilled artisan and the step of comparing the expression pattern can be performed by a second skilled artisan.
  • the present invention provides a method of diagnosing cancer comprising: a) matching a test transposable element expression pattern with a known expression pattern for a type of cancer; and b) diagnosing the type of cancer based on matching of the test expression pattern with a known expression pattern for a type of cancer.
  • one of skill in the art can obtain an ovarian tumor cell and determine the expression pattern of one or more transposable element families. By determining which transposable element families are expressed as well as which members of these transposable element families are expressed, one of skill in the art can assign this pattern to an ovarian tumor cell. This can be done for an ovarian tumor cell at different stages of cancer, such that a library of expression patterns are readily available to not only diagnose but stage ovarian cancer.
  • cancer cell such as a carcinoma cell, a fibroma cell, a sarcoma cell, a teratoma cell, a blastoma cell, a breast tumor cell of epithelial origin, an ovarian tumor cell of epithelial, stromal or germ cell origin, mixed cell types from a tumor or any other cancer cell.
  • a carcinoma cell such as a carcinoma cell, a fibroma cell, a sarcoma cell, a teratoma cell, a blastoma cell, a breast tumor cell of epithelial origin, an ovarian tumor cell of epithelial, stromal or germ cell origin, mixed cell types from a tumor or any other cancer cell.
  • Such libraries of expression patterns are useful for diagnosis, staging and treatment.
  • a sample can be obtained from a patient or subject in need of diagnosis and assayed for transposable element expression. Once the expression pattern is determined according to the methods of the present invention, this expression pattern can be compared to a library of expression patterns to determine the type of cancer as well as the stage of cancer associated with the expression pattern. Once this is determined, appropriate treatment can be prescribed.
  • the present methods are also useful for identifying expression patterns of cancer cells after therapeutic intervention.
  • a sample can be obtained from a patient or subject undergoing treatment for a cancer such as prostate cancer, lymphoma, skin cancer, GI-tract cancer or any other type of cancer.
  • Expression patterns can be obtained and compared to expression patterns before treatment. In this way, the changes in transposable element expression can be monitored such that one of skill in the art would know which transposable element families as well as which members of each family are affected by the treatment. If improvement is seen in the patient, these improvements can be attributed to changes in transposable element expression. Since the skilled artisan will have reference patterns for a normal tissue or cell, changes in transposable element expression after treatment can be monitored to determine if the treatment results in a transposable element expression pattern that more closely resembles normal or “baseline” expression patterns. Improvements can also be monitored clinically by observing changes in tissue health, cellular changes and changes in the subject's overall health. In this way, one of skill in the art can correlate clinical changes with changes in transposable element expression.
  • tissue sample For cancers such as breast cancer and ovarian cancer, once a tissue sample is obtained from a subject, this tissue sample can be compared to a library of tissue samples from many subjects, representing various stages of the cancerous tumor.
  • tissue sample By comparing the tissue sample to a library of tissue samples with known transposable element expression patterns, one of skill in the art can tailor treatment to the individual needs of the subject. For example, if the expression pattern for the subject matches the expression pattern of a particular stage of cancer that is amenable to treatment with a chemotherapeutic agent, then the subject is a candidate for that treatment.
  • one of skill in the art can determine the likelihood that the subject will respond to a particular treatment by determining whether or not the subject's pattern corresponds to patterns obtained for those who have responded to treatment.
  • the patterns in the libraries utilized for comparison purposes can be grouped by age, medical history or other categories in order to better determine the likelihood of response for subjects.
  • the pattern obtained from the subject may correspond to a pattern for a stage of cancer that does not respond to any available treatment. In cases such as these,.one of skill in the art may determine that treatment may not be advisable because the subject may suffer unnecessarily with little or no likelihood of success.
  • the present invention also provides a method of determining the effectiveness of an anti-cancer therapeutic in a subject comprising: a) determining expression of one or more families of transposable elements, in a sample obtained from the subject, to obtain a first expression pattern; b) administering an anti-cancer therapeutic to the subject; c) determining expression of one or more families of transposable elements in a sample obtained from the subject after administration of an anti-cancer therapeutic to obtain a second expression pattern; and d) comparing the second expression pattern with the first expression pattern such that if the differences between the expression patterns can be correlated with successful treatment, the anti-cancer therapeutic is an effective anti-cancer therapeutic.
  • the changes observed between expression patterns can vary depending on the type of cancer and the stage of cancer. The changes observed can also vary depending on the size, age, weight and other physiological characteristics of the subject.
  • an effective anti-cancer therapeutic will result in fewer transposable elements being expressed in the second expression pattern as compared to the first expression pattern. In other instances, there may be more transposable elements expressed in the second pattern as compared to the first expression pattern.
  • one of skill in the art can diagnose a cancer utilizing the methods of the present invention and assign a first expression pattern to a sample from a subject. The following example is not meant to be limiting and the numbering of transposable elements appears for illustrative purposes only and not for purposes of identifying any particular retroelement sequences.
  • the first expression pattern comprises the expression of transposable elements 1, 3, 5, 7, 9 from transposable element family A, the expression of transposable elements 23, 56 and 78 from transposable element family B and the expression of transposable elements 10, 15, 25 from transposable element family C.
  • a second expression pattern is obtained.
  • the second expression pattern comprises, for example, the expression of transposable elements 3, 5, 9 from family A, the expression of transposable element 23 from family B and the expression of transposable element 15 from transposable element family C.
  • transposable elements 1 and 7 from family A
  • transposable elements 56 and 78 from family B
  • transposable elements 10 and 25 from transposable element family C.
  • the skilled artisan can continue to monitor changes throughout treatment in order to determine which transposable elements are suppressed or expressed as treatment progresses.
  • One of skill in the art can also compare the expression pattern obtained after treatment to the expression pattern of a normal, non-cancerous cell to determine how the treatment is progressing.
  • the treatment can be said to be successful, however, the expression pattern need not be exactly like the expression pattern of a normal cell in order to deem a treatment effective. In effect, if the changes in transposable element expression after treatment are indicative of progression toward the expression pattern of a normal cell, the treatment can be said to be successful.
  • the present invention also provides methods of assessing the methylation status of transposable element sequences and its role in cancer development and progression.
  • the present invention also provides methods for the determination of methylation patterns of transposable element sequences.
  • These transposable element methylation patterns can be used in combination with transposable element expression patterns described herein to diagnose, classify and stage cancer.
  • Also provided by the present invention is a method of determining a methylation pattern of one or more families of transposable elements genes in a sample comprising determining methylation of one or more families of transposable elements.
  • methylation patterns can include one, two, three, four, five, six, seven, eight, nine, ten, twenty or more families of transposable elements and at least one, two, three, four, five, ten, fifteen, twenty, twenty-five, fifty, one hundred, two hundred, three hundred, four hundred, five hundred members, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members of each transposable element family.
  • the present invention provides for the determination of a methylation pattern of one family of transposable elements in which one, two, three, four, five, ten, fifteen, twenty, twenty five, fifty, one hundred, two hundred, three hundred, four hundred, five hundred members, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members of the transposable element family are analyzed.
  • the present invention also provides for the determination of a methylation pattern of two families, wherein one, two, three, four, five, ten, fifteen, twenty, twenty five, fifty, one hundred, two hundred, three hundred, four hundred, five hundred members, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members are analyzed for each family.
  • the invention provides for the determination of a methylation pattern of three families, wherein one, two, three, four, five, ten, fifteen, twenty, twenty five fifty, one hundred, two hundred, three hundred, four hundred, five hundred members, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members are analyzed for each family.
  • the invention provides for the determination of a methylation pattern of multiple families, for example, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 families wherein one, two, three, four, five, ten, fifteen, twenty, twenty five fifty, one hundred, two hundred, three hundred, four hundred, five hundred, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members are analyzed for each family.
  • families for example, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 families wherein one, two, three, four, five, ten, fifteen, twenty, twenty five fifty, one hundred, two hundred, three hundred, four hundred, five hundred, one thousand, two thousand, three thousand, four thousand, five thousand, six
  • the present invention provides a method of assigning a methylation pattern of transposable elements to a type of cancerous cell in a sample, comprising: determining the methylation pattern of one or more families of transposable elements; and assigning the methylation pattern obtained from step a) to the type of cancerous cell in the sample.
  • the present invention also provides a method of diagnosing cancer comprising: a) determining the methylation pattern of one or more families of transposable elements in a sample to obtain a methylation pattern; b) matching the methylation pattern of step a) with a known methylation pattern for a type of cancer; and c) diagnosing the type of cancer based on matching of the methylation pattern of a) with a known methylation pattern for a type of cancer.
  • the methylation pattern obtained from a sample taken from a subject can be obtained from outside sources, such as a testing laboratory or a commercial source. Therefore, the step of obtaining the methylation pattern can be performed by one skilled artisan and the step of comparing the methylation pattern can be performed by a second skilled artisan.
  • the present invention provides a method of diagnosing cancer comprising: a) matching a test transposable element methylation pattern with a known methylation pattern for a type of cancer; and b) diagnosing the type of cancer based on matching of the test methylation pattern with a known methylation pattern for a type of cancer.
  • one of skill in the art can obtain an ovarian cancer sample and determine the methylation pattern of one or more transposable element families. By determining which transposable element families are methylated as well as which members of these transposable element families are methylated, one of skill in the art can assign this methylation pattern to an ovarian cancer sample. This can be done for ovarian cancer samples at different stages of cancer, such that a library of methylation patterns are readily available to not only diagnose but stage ovarian cancer.
  • cancer cell such as a carcinoma cell, a fibroma cell, a sarcoma cell, a teratoma cell, a blastoma cell, a breast tumor cell of epithelial origin, an ovarian tumor cell of epithelial, stromal or germ cell origin, mixed cell types from a tumor or any other cancer cell.
  • a carcinoma cell such as a carcinoma cell, a fibroma cell, a sarcoma cell, a teratoma cell, a blastoma cell, a breast tumor cell of epithelial origin, an ovarian tumor cell of epithelial, stromal or germ cell origin, mixed cell types from a tumor or any other cancer cell.
  • Such libraries of expression patterns are useful for diagnosis, staging and treatment.
  • a sample can be obtained from a patient or subject in need of diagnosis and assayed for transposable element methylation.
  • this methylation pattern can be compared to a library of methylation patterns to determine the type of cancer as well as the stage of cancer associated with the methylation pattern. Once this is determined, appropriate treatment can be prescribed.
  • the present methods are also useful for identifying methylation patterns of cancer cells after therapeutic intervention.
  • a sample can be obtained from a patient or subject undergoing treatment for a cancer such as prostate cancer, lymphoma, skin cancer, GI-tract cancer or any other type of cancer.
  • Methylation patterns can be obtained and compared to methylation patterns before treatment. In this way, the changes in transposable element methylation can be monitored such that one of skill in the art would know which transposable element families as well as which members of each family are affected by the treatment. If improvement is seen in the patient, these improvements can be attributed to changes in transposable element methylation.
  • transposable element methylation after treatment can be monitored to determine if the treatment results in a transposable element methylation pattern that more closely resembles normal or “baseline” methylation patterns. Improvements can also be monitored clinically by observing changes in tissue health, cellular changes and changes in the subject's overall health. In this way, one of skill in the art can correlate clinical changes with changes in transposable element methylation.
  • tissue sample For cancers such as breast cancer and ovarian cancer, once a tissue sample is obtained from a subject, this tissue sample can be compared to a library of tissue samples from many subjects, representing various stages of the cancerous tumor.
  • tissue sample By comparing the tissue sample to a library of tissue samples with known transposable element methylation patterns, one of skill in the art can tailor treatment to the individual needs of the subject. For example, if the methylation pattern for the subject matches the methylation pattern of a particular stage of cancer that is amenable to treatment with a chemotherapeutic agent, then the subject is a candidate for that treatment.
  • one of skill in the art can determine the likelihood that the subject will respond to a particular treatment by determining whether or not the subject's pattern corresponds to patterns obtained for those who have responded to treatment. In this way, treatments can be personalized to maximize the outcome while minimizing unnecessary side effects.
  • the patterns in the libraries utilized for comparison purposes can be grouped by age, medical history or other categories in order to better determine the likelihood of response for subjects.
  • the pattern obtained from the subject may correspond to a pattern for a stage of cancer that does not respond to any available treatment. In cases, such as these, one of skill in the art may determine that treatment may not be advisable because the subject may suffer unnecessarily with little or no likelihood of success.
  • a treatment is not successful in a particular subject, the skilled artisan will recognize this by noting that the methylation pattern is not changing as expected, i.e., the methylation pattern is not changing such that the methylation pattern more closely resembles the methylation pattern of a noncancerous or successfully treated cancer cell, and other dosages, therapies or treatments can be employed.
  • the present invention also provides a method of determining the effectiveness of an anti-cancer therapeutic in a subject comprising: a) determining the methylation pattern of one or more families of transposable elements, in a sample obtained from the subject, to obtain a first methylation pattern; b) administering an anti-cancer therapeutic to the subject; c) determining the methylation pattern of one or more families of transposable elements in a sample obtained from the subject after administration of an anti-cancer therapeutic to obtain a second methylation pattern; and d) comparing the second methylation pattern with the first methylation pattern such that if the differences between the methylation patterns can be correlated with successful treatment, the anti-cancer therapeutic is an effective anti-cancer therapeutic.
  • the changes observed between methylation patterns can vary depending on the type of cancer and the stage of cancer.
  • the changes in methylation patterns can also vary based on the size, age, weight and other physiological characteristics of the subject.
  • an effective anti-cancer therapeutic will result in fewer transposable elements being methylated in the second methylation pattern as compared to the first methylation pattern. In other instances, there may be more transposable elements methylated in the second pattern as compared to the first methylation pattern.
  • one of skill in the art can diagnose a cancer utilizing the methods of the present invention and assign a first methylation pattern to a sample from a subject. The following example is not meant to be limiting and the numbering of transposable elements appears for illustrative purposes only and not for purposes of identifying any particular retroelement sequences.
  • this first methylation pattern comprises the methylation of transposable elements 2, 4, 6, 8 and 10 from transposable element family A, the methylation of transposable elements 24, 57 and 79 from transposable element family B and the methylation of transposable elements 11, 16, and 26 from transposable element family C.
  • a second methylation pattern is obtained.
  • the second expression pattern comprises, for example, the methylation of transposable elements 2, 4, 6, 8, 10, 12 and 14 from family A, the methylation of transposable element 24, 57, 79 and 80 from family B and the methylation of transposable elements 11, 16, 26 and 32 from transposable element family C.
  • This second methylation pattern can be compared to the methylation pattern of a normal cell to see if the treatment is progressing toward a methylation pattern associated with a non-cancerous cell.
  • This second methylation pattern can also be compared to methylation patterns for different stages of the particular cancer being treated in order to determine if this pattern corresponds to an improvement or a deterioration in the subject's condition.
  • the skilled artisan can continue to monitor changes throughout treatment in order to determine which transposable elements are methylated or non-methylated, and whether or not an improvement can be correlated to changes in methylation, as treatment progresses.
  • the methylation state of non-cancerous cells can serve as a guide to one of skill in the art in determining the effectiveness of a treatment.
  • One of skill in the art can compare the methylation pattern obtained after treatment to the methylation pattern of a normal, non-cancerous cell to determine how the treatment is progressing. If the methylation pattern after treatment resembles the methylation pattern of a normal cell, the treatment can be said to be successful, however, the methylation pattern need not be exactly like the methylation pattern of a normal cell in order to deem a treatment effective. In other words, if the changes in transposable element sequence methylation after treatment are indicative of progression toward the methylation pattern of a normal cell, the treatment can be said to be successful.
  • the methylation patterns of the present invention can be correlated to transposable element expression patterns and/or chromatin status patterns described herein, such that one of skill in the art, upon obtaining a particular expression pattern and/or a chromatin status pattern, will also know what the methylation status of the sample is. Also, upon obtaining upon obtaining a particular methylation pattern, one of skill in the art will also know the expression pattern and/or chromatin status of the sample.
  • Methods of measuring methylation include, but are not limited to methylation-specific PCR, methylation microarray analysis and ChIP (a chromatin immunoprecipitation approach) analysis. Methylation can also be monitored by digestion of nucleic acid sequences with methylation sensitive and non-sensitive restriction enzymes followed by Southern blotting or PCR analysis of the restriction products (See Takai et al. “Hypomethylation of LINE1 retrotransposon in human hepatocellular carcinomas, but not in surrounding liver cirrhosis” Jpn J. Clin. Oncol. 30(7) 306-309).
  • One of skill in the art could also utilize methods in which genomic DNA is digested followed by PCR. (See, for example, Cartwright et al., “Analysis of Drosophila chromatin structure in vivo” Methods in Enzymology, Vol. 304)
  • Methylation-specific PCR (MSP) technology utilizes the fact that DNA in humans is methylated mainly at certain cytosines located 5′ to guanosine. This occurs especially in, GC-rich regions, known as CpG islands. To distinguish the methylation state of a sequence, MSP relies on differential chemical modification of cytosine residues in DNA. Treatment with sodium bisulfite converts unmethylated cytosine residues into uracil, leaving the methylated cytosines unchanged. This modification thus creates different DNA sequences for methylated and unmethylated DNA. PCR primers can then be designed so as to distinguish between these different sequences.
  • Two sets of primers are designed: one set with sequences annealing to unchanged (methylated in the genomic DNA) cytosines and the other set with sequences annealing to the altered (unmethylated in the genomic DNA) cytosines.
  • a comparison of PCR results using the two sets of primers reveals the methylation state of a PCR product. If the primer set with the altered sequence gives a PCR product, then the indicated cytosine was unmethylated. If the primer set with the unchanged sequence gives a PCR product, then the cytosines were methylated and thus protected from alteration.
  • Evron et al. (“Detection of breast cancer cells in ductal lavage fluid by methylation-specific PCR,” Lancet 2001, 357: 1335-1336) describes the use of MSP to detect breast cancer and is hereby incorporated in its entirety by this reference.
  • transposable element array To use a microarray to study transposable element methylation, one of skill in the art would select for methylated and unmethylated DNA from total genomic DNA. The selectively isolated DNA is then hybridized to the transposable element array either directly or after amplification and patterns between various cell types / tissue types as described earlier in the patent application.
  • chromatin immunoprecipitation (CHIP ).
  • Another method utilizes a column binding approach and a third method involves ligation of adapters to fragmented genomic DNA and methylation-specific restriction digestion of the ligation products followed by PCR amplification.
  • the selected DNA fragments are labeled by incorporation of dNTPs coupled with fluorescent dyes (for example Cy3 or Cy5 coupled dNTPs) and hybridization to the microarray is performed according to standard protocols.
  • fluorescent dyes for example Cy3 or Cy5 coupled dNTPs
  • One of skill in the art could utilize the BioPrime DNA labeling system from Life Technologies or other kits available for such labeling.
  • microarray techniques would be known to one of skill in the art.
  • U.S. Pat. No. 6,410,229 and U.S. Pat. No. 6,344,316 both hereby incorporated by this reference, describe methods of hybridizing nucleic acids to high density nucleic acid arrays.
  • one skilled in the art would first produce fluorescent-labeled DNA isolated from the tissue of interest.
  • a batch of labeled genomic/amplified genomic DNAs representing either one sample or a mixture of two samples from the tissue sources of interest is added to an array of oligonucleotides representing a plurality of known transposable elements, as described above, under conditions that result in hybridization of the DNAs to complementary-sequence oligonucleotides in the array.
  • the array is then examined by fluorescence under fluorescence excitation conditions in which transposable element oligonucleotides in the array that are hybridized to genomic/amplified genomic DNAs derived from the tissue of interest can be detected and quantified.
  • ChIP technology involves in vivo formaldehyde cross-linking of DNA and associated proteins in intact cells, followed by selective immunoprecipitation of protein-DNA complexes with specific antibodies. Such an approach allows detection of any protein at its in vivo binding site directly. In particular, proteins that are not bound directly to DNA or that depend on other proteins for binding activity in vivo can be analyzed by this method.
  • methylation complexes can be cross-linked to transposable element sequences to which they are bound and then an antibody specific to one of the proteins (i.e., one of the proteins involved in the methylation complex, such as methyltransferase or a protein having a methyl binding site, for example, MBD1) can be utilized to immunoprecipitate the methylation complex-DNA bound sequence.
  • the complex can then be chemically released and the transposable element sequence to which it was bound can be identified.
  • the column binding approach is used to select for methylated DNA after genomic DNA extraction.
  • the column contains methyl-CpG-binding proteins, for example the methyl-binding domain of rat MeCP2, covalently linked to a histidine tag, then attached to a Ni-agarose matrix.
  • Fragmented genomic DNA digested with restriction enzymes, for example Mse1 is run through the column.
  • the column retains DNA containing methylated cytosines, unmethylated DNA is collected from the flow-through. Retained methylated DNA is recovered from the column.
  • Formaldehyde crosslinking followed by chromatin immunoprecipitation is reviewed in Orlando 2000.
  • DNA and nearby proteins are cross-linked in vivo, followed by sonication of the tissue/cell suspension.
  • the DNA is fragmented in the process.
  • Antibodies recognizing methyl-binding proteins are added and the immune complexes are collected, thereby precipitating methylated DNA with associated proteins.
  • DNA without methyl-binding proteins will be collected from the supernatant.
  • the cross-linking step is then reversed for both fractions, followed by a DNA purification step.
  • the isolated DNA can be ligated to linker oligonucleotides and amplified by PCP, Fluorescence labeling and hybridization is then performed as described above.
  • Linker ligation/Methylation-specific restriction/ PCR can also be utilized.
  • the methods of the present invention can utilize a modified version of DMH (Differential Methylation Hybridization) (References: Huang et al. ‘Methylation profiling of CpG islands in human breast cancer cells’ Human Molecular Genetics 1999, Vol. 8, No. 3 and Yan et al. ‘Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays’ Cancer Research 2001, 61, 8375-8380).
  • Genomic DNA is digested with MseI. Then, the ends of the resulting fragments are ligated to linker oligonucleotides.
  • Ligated fragments undergo restriction digestion with methylation-sensitive enzymes BstUI and/or HpaII, followed by PCR amplification of undigested fragments. Fluorescence labeling and hybridization is then performed as described above.
  • a COT-1 subtractive hybridization step can be utilized at some point before labeling the DNA to separate out the highly repetitive sequences from the sample (See Craig et al. ‘Removal of repetitive sequences from FISH probes using PCR-assisted affinity chromatography’ Human Genetics 1997, Vol. 100, 472-476).
  • methylation-specific oligonucleotide (MSO) microarray uses bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest.
  • the amplified product therefore, may contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status.
  • a test sample is hybridized to a set of oligonucleotide arrays that discriminate between methylated and unmethylated cytosine at specific nucleotide positions, and quantitative differences in hybridization are determined by fluorescence analysis.
  • methylation micro array techniques see Gitan et al.
  • the present invention also provides methods of assessing the chromatin status of transposable element sequences and its role in cancer development and progression.
  • the present invention also provides methods for the determination of chromatin status patterns of transposable element sequences.
  • chromatin status patterns By analyzing global chromatin status patterns of transposable element sequences and transposable element families, one of skill in the art can assign particular transposable element chromatin status patterns to types of cancer. Such chromatin status patterns can be used to diagnose, classify and stage cancer.
  • These transposable element chromatin status patterns can be used in combination with transposable element expression patterns and/or methylation patterns described herein to diagnose, classify and stage cancer.
  • chromatin status refers to the chromosomal structure or the chromosomal accessibility or the ability of restriction enzymes to access a transposable element sequence or a fragment thereof Therefore, chromatin status patterns can contain sequences that are accessible to restriction enzymes and sequences that are not accessible to restriction enzymes.
  • Also provided by the present invention is a method of determining a chromatin status pattern of one or more families of transposable element genes in a sample comprising determining chromatin status of one or more families of transposable elements.
  • chromatin status patterns can include one, two, three, four, five, six, seven, eight, nine, ten, twenty or more families of transposable elements and at least one, two, three, four, five, ten, fifteen, twenty, twenty-five, fifty, one hundred, two hundred, three hundred, four hundred, five hundred members, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members of each transposable element family.
  • the present invention provides for the determination of a chromatin status pattern of one family of transposable elements in which one, two, three, four, five, ten, fifteen, twenty, twenty five fifty, one hundred, two hundred, three hundred, four hundred, five hundred members, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members of the transposable element family are analyzed.
  • the present invention also provides for the determination of a chromatin status pattern of two families, wherein one, two, three, four, five, ten, fifteen, twenty, twenty five fifty, one hundred, two hundred, three hundred, four hundred, five hundred members, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members are analyzed for each family.
  • the invention provides for the determination of a chromatin status pattern of three families, wherein one, two, three, four, five, ten, fifteen, twenty, twenty five fifty, one hundred, two hundred, three hundred, four hundred, five hundred members, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members are analyzed for each family.
  • the invention provides for the determination of a chromatin status pattern of multiple families, for example, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 families wherein one, two, three, four, five, ten, fifteen, twenty, twenty five fifty, one hundred, two hundred, three hundred, four hundred, five hundred, one thousand, two thousand, three thousand, four thousand, five thousand, six thousand, seven thousand, eight thousand, nine thousand, ten thousand, twenty thousand, fifty thousand, one hundred thousand, two hundred thousand, three hundred thousand, four hundred thousand or five hundred thousand members are analyzed for each family.
  • families for example, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 families wherein one, two, three, four, five, ten, fifteen, twenty, twenty five fifty, one hundred, two hundred, three hundred, four hundred, five hundred, one thousand, two thousand, three thousand, four thousand, five thousand,
  • the present invention provides a method of assigning a chromatin status pattern of transposable elements to a type of cancerous cell in a sample, comprising: determining the chromatin status pattern of one or more families of transposable elements; and assigning the chromatin status pattern obtained from step a) to the type of cancerous cell in the sample.
  • the present invention also provides a method of diagnosing cancer comprising: a) determining the chromatin status pattern of one or more families of transposable elements in a sample to obtain a chromatin status pattern; b) matching the chromatin status pattern of step a) with a known chromatin status pattern for a type of cancer; and c) diagnosing the type of cancer based on matching of the chromatin status pattern of a) with a known chromatin status pattern for a type of cancer.
  • the chromatin status pattern obtained from a sample taken from a subject can be obtained from outside sources, such as a testing laboratory or a commercial source. Therefore, the step of obtaining the chromatin status pattern can be performed by one skilled artisan and the step of comparing the chromatin status pattern can be performed by a second skilled artisan.
  • the present invention provides a method of diagnosing cancer comprising: a) matching a test transposable element chromatin status pattern with a known chromatin status pattern for a type of cancer; and b) diagnosing the type of cancer based on matching of the test chromatin status pattern with a known chromatin status pattern for a type of cancer.
  • one of skill in the art can obtain an ovarian cancer sample and determine the chromatin status pattern of one or more transposable element families. By determining the chromosomal accessibility of transposable element families as well as the chromosomal accessibility of members of these transposable element families, one of skill in the art can assign this chromatin status pattern to an ovarian cancer sample. This can be done for ovarian cancer samples at different stages of cancer, such that a library of chromatin status patterns are readily available to not only diagnose but stage ovarian cancer.
  • cancer cell such as a carcinoma cell, a fibroma cell, a sarcoma cell, a teratoma cell, a blastoma cell, a breast tumor cell of epithelial origin, an ovarian tumor cell of epithelial, stromal or germ cell origin, mixed cell types from a tumor or any other cancer cell.
  • a carcinoma cell such as a carcinoma cell, a fibroma cell, a sarcoma cell, a teratoma cell, a blastoma cell, a breast tumor cell of epithelial origin, an ovarian tumor cell of epithelial, stromal or germ cell origin, mixed cell types from a tumor or any other cancer cell.
  • Such libraries of expression patterns are useful for diagnosis, staging and treatment.
  • a sample can be obtained from a patient or subject in need of diagnosis and assayed for chromatin status.
  • this chromatin status pattern can be compared to a library of chromatin status patterns to determine the type of cancer as well as the stage of cancer associated with the chromatin pattern. Once this is determined, appropriate treatment can be prescribed.
  • the present methods are also useful for identifying chromatin status patterns of cancer cells after therapeutic intervention.
  • a sample can be obtained from a patient or subject undergoing treatment for a cancer such as prostate cancer, lymphoma, skin cancer, GI-tract cancer or any other type of cancer.
  • Chromatin status patterns can be obtained and compared to chromatin status patterns before treatment. In this way, the changes in transposable element chromatin status can be monitored such that one of skill in the art would know which transposable element families as well as which members of each family are affected by the treatment. If improvement is seen in the patient, these improvements can be attributed to changes in transposable element chromatin status.
  • transposable element chromatin status after treatment can be monitored to determine if the treatment results in a transposable element chromatin status pattern that more closely resembles normal or “baseline” chromatin status patterns. Improvements can also be monitored clinically by observing changes in tissue health, cellular changes and changes in the subject's overall health. In this way, one of skill in the art can correlate clinical changes with changes in transposable element chromatin status.
  • tissue sample For cancers such as breast cancer and ovarian cancer, once a tissue sample is obtained from a subject, this tissue sample can be compared to a library of tissue samples from many subjects, representing various stages of the cancerous tumor.
  • tissue sample By comparing the tissue sample to a library of tissue samples with known transposable element chromatin status patterns, one of skill in the art can tailor treatment to the individual needs of the subject. For example, if the chromatin status pattern for the subject matches the chromatin status pattern of a particular stage of cancer that is amenable to treatment with a chemotherapeutic agent, then the subject is a candidate for that treatment.
  • one of skill in the art can determine the likelihood that the subject will respond to a particular treatment by determining whether or not the subject's pattern corresponds to patterns obtained for those who have responded to treatment. In this way, treatments can be personalized to maximize the outcome while minimizing unnecessary side effects.
  • the patterns in the libraries utilized for comparison purposes can be grouped by age, medical history or other categories in order to better determine the likelihood of response for subjects.
  • the pattern obtained from the subject may correspond to a pattern for a stage of cancer that does not respond to any available treatment. In cases, such as these, one of skill in the art may determine that treatment may not be advisable because the subject may suffer unnecessarily with little or no likelihood of success.
  • effective treatments may involve decreasing the chromatin accessibility of certain transposable elements and increasing the chromatin accessibility of others. Therefore, once libraries of chromatin status patterns are established from untreated and treated cancer subjects, one of skill in the art will know whether or not treatment is effective in a particular subject by comparing the chromatin status pattern of a sample from the patient at different stages of treatment, with reference patterns established for the successful treatment of that particular type of cancer.
  • chromatin status pattern is not changing as expected, i.e., the chromatin status pattern is not changing such that the chromatin status pattern more closely resembles the chromatin status pattern of a non-cancerous or successfully treated cancer cell, and other dosages, therapies or treatments can be employed.
  • the present invention also provides a method of determining the effectiveness of an anti-cancer therapeutic in a subject comprising: a) determining the chromatin status pattern of one or-more families of transposable elements, in a sample obtained from the subject, to obtain a first chromatin status pattern; b) administering an anti-cancer therapeutic to the subject; c) determining the chromatin status pattern of one or more families of transposable elements in a sample obtained from the subject after administration of an anti-cancer therapeutic to obtain a second chromatin status pattern; and d) comparing the second chromatin status pattern with the first chromatin status pattern such that if the differences between the chromatin status patterns can be correlated with successful treatment, the anti-cancer therapeutic is an effective anti-cancer therapeutic.
  • the changes observed between chromatin status patterns can vary depending on the type of cancer and the stage of cancer.
  • the changes in chromatin status patterns can also vary based on the size, age, weight and other physiological characteristics of the subject.
  • an effective anti-cancer therapeutic will result in fewer transposable elements being accessible to restriction enzymes in the second chromatin status pattern as compared to the first chromatin status pattern. In other instances, there may be more transposable elements accessible to restriction enzymes in the second pattern as compared to the first chromatin status pattern.
  • one of skill in the art can diagnose a cancer utilizing the methods of the present invention and assign a first chromatin status pattern to a sample from a subject. The following example is not meant to be limiting and the numbering of transposable elements appears for illustrative purposes only and not for purposes of identifying any particular transposable element-sequences.
  • this first chromatin status pattern comprises the chromatin status of transposable elements 2 (accessible), 4 (not accessible), 6 (accessible), 8 (not accessible) and 10 (not accessible) from transposable element family A, the chromatin status of transposable elements 24 (not accessible), 57 (accessible) and 79 (not accessible) from transposable element family B and the chromatin status of transposable elements 11 (not accessible), 16 (accessible), and 26 (not accessible) from transposable element family C.
  • a second chromatin status pattern is obtained.
  • the second chromatin status pattern comprises, for example, the chromatin status of transposable elements 2 (not accessible), 4 (not accessible), 6 (accessible), 8 (not accessible) and 10 (not accessible) from family A, the chromatin status of transposable element 24 (not accessible), 57 (not accessible) and 79 (accessible) from family B and the chromatin status of transposable elements 11 (accessible), 16 (not accessible) and 26 (not accessible) from transposable element family C.
  • the skilled artisan upon comparing the patterns, will determine that the anti-cancer therapeutic results in changes in the chromatin status of transposable element 2 from family A, transposable elements 57 and 79 from family B, and transposable element 11 from transposable element family C.
  • This second chromatin status pattern can be compared to the chromatin status pattern of a normal cell to see if the treatment is progressing toward a chromatin status pattern associated with a non-cancerous cell.
  • This second chromatin status pattern can also be compared to chromatin status patterns for different stages of the particular cancer being treated in order to determine if this pattern corresponds to an improvement or a deterioration in the subject's condition.
  • the skilled artisan can continue to monitor changes throughout treatment in order to determine which transposable elements are accessible or not accessible and whether or not an improvement can be correlated to changes in chromatin status, as treatment progresses.
  • the chromatin status state of non-cancerous cells can serve as a guide to one of skill in the art in determining the effectiveness of a treatment.
  • One of skill in the art can compare the chromatin status pattern obtained after treatment to the chromatin status pattern of a normal, non-cancerous cell to determine how the treatment is progressing. If the chromatin status pattern after treatment resembles the chromatin status pattern of a normal cell, the treatment can be said to be successful, however, the chromatin status pattern need not be exactly like the chromatin status pattern of a normal cell in order to deem a treatment effective. In other words, if the changes in transposable element sequence chromatin status after treatment are indicative of progression toward the chromatin status pattern of a normal cell, the treatment can be said to be successful.
  • the chromatin status patterns of the present invention can be correlated to transposable element expression patterns and/or methylation patterns described herein, such that one of skill in the art, upon obtaining a particular expression pattern and/or methylation pattern, will also know what the chromatin status of the sample is. Also, upon obtaining a particular chromatin status pattern, one of skill in the art will also know the expression pattern and/or methylation pattern of the sample.
  • the methods of the present invention can also be utilized to differentiate between subtypes of cancers.
  • mantle cell lymphoma and grades I/II follicular lymphoma are subtypes of non-Hodgkin's lymphoma.
  • adenocarcinoma, large cell carcinoma, spindle cell carcinoma, squamous cell carcinoma, adenosquamous carcinoma and small cell carcinoma are all subtypes of lung cancer.
  • Numerous subtypes for other cancers are also known and they can be differentiated by the methods of the present invention.
  • the skilled artisan can make a more accurate diagnosis of a particular type of cancer.
  • the differences in the expression patterns, chromatin status and methylation patterns of the transposable element sequences allows the skilled artisan to differentiate between subtypes and thus better stage the cancer as well as administer treatment best suited for a specific cancer subtype.
  • the present invention also provides a computer system comprising a) a database including records comprising a plurality of reference retroelement expression patterns, and associated diagnosis and therapy data; and b) a user interface capable of receiving a selection of one or more test retroelement expression patterns for use in determining matches between a test retroelement expression pattern and a reference retroelement expression pattern, and displaying the records associated with matching expression patterns.
  • the computer systems of the present invention can also include a database including records comprising a plurality of reference methylation patterns, and associated diagnosis and therapy data, b) a user interface capable of receiving a selection of one or more test methylation patterns for use in determining matches between a test methylation pattern and the reference methylation pattern, and displaying the records associated with matching expression patterns.
  • a computer system comprising a) a database including records comprising a plurality of reference chromatin status patterns, and associated diagnosis and therapy data; and b) a user interface capable of receiving a selection of one or more test chromatin status patterns for use in determining matches between a test chromatin status pattern and a reference chromatin status pattern, and displaying the records associated with matching expression patterns.
  • expression patterns, methylation patterns and/or chromatin status patterns identified from subjects can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer.
  • the words “recorded” and “stored” refer to a process for storing information on a computer medium.
  • a skilled artisan can readily adopt any of the presently known methods for recording information on a computer readable medium to generate a list of sequences comprising one or more of the nucleic acids of the invention.
  • Another aspect of the present invention is a computer readable medium having recorded thereon at least 2, 5, 10, 15, 20, 25, 30, 50, 100, 200, 250, 300, 400, 500, 1000, 2000, 3000, 4000 or 5000 expression patterns, methylation patterns and/or chromatin status patterns of the invention or patterns identified from subjects.
  • Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media.
  • the computer readable media may be a hard disc, a floppy disc, a magnetic tape, CD-ROM, DVD, RAM, or ROM as well as other types of other-media known to those skilled in the art.
  • Embodiments of the present invention include systems, particularly computer systems which contain the sequence information described herein.
  • a computer system refers to the hardware components, software components, and data storage components used to store and/or analyze the expression patterns of the present invention or other expression patterns.
  • the computer system preferably includes the computer readable media described above, and a processor for accessing and manipulating the data.
  • the computer is a general purpose system that comprises a central processing unit (CPU), one or more data storage components for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components.
  • CPU central processing unit
  • data storage components for storing data
  • data retrieving devices for retrieving the data stored on the data storage components.
  • the computer system includes a processor connected to a bus which is connected to a main memory, preferably implemented as RAM, and one or more data storage devices, such as a hard drive and/or other computer readable media having data recorded thereon.
  • the computer system further includes one or more data retrieving devices for reading the data stored on the data storage components.
  • the data retrieving device may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, a hard disk drive, a CD-ROM drive, a DVD drive, etc.
  • the data storage component is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded thereon.
  • the computer system may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving device.
  • the computer system may further comprise an expression pattern comparer for comparing the expression pattern(s) stored on a computer readable medium to expression pattern(s) stored on a computer readable medium.
  • An “expression pattern comparer” refers to one or more programs which are implemented on the computer system to compare a nucleotide sequence with other nucleotide sequences. Similarly, programs capable of comparing methylation status patterns and chromatin status patterns are also contemplated by the present invention.
  • This invention also provides for a computer program that correlates expression patterns with a particular stage of cancer. Similarly, the present invention also provides a computer program that correlates methylation patterns with a particular stage of cancer. Also provided is a computer program that correlates chromatin status with a particular stage of cancer.
  • the computer programs of this invention can optionally include treatment options or drug indications for subjects with expression patterns associated with cancer or the risk of developing cancer.
  • FIG. 1 shows the upregulation of HERV-K and HERV-W families in a cancer sample, compared with a non-cancer sample.
  • Methylation levels of HERV-W, and L1 were compared among different ovarian samples.
  • Ten micrograms of genomic DNA were digested either with a methylation sensitive restriction enzyme (HpaII) or with its methylation insensitive isoschizomer (MspI). These enzymes recognize the palindromic sequence CCGG, which is found in diverse positions in the promoter regions of these retroelements. Digestion is carried out overnight at 37° C. with 10 to 16 excess of needed enzyme to ensure complete digestion of the DNA. A control for DNase contamination is included by incubating the same amount of DNA with buffer and water without the enzyme. Digested DNA is run on an agarose gel and transferred to a nylon membrane with NaOH. Membranes are then prehybridized for 1 hour with 10 mg of herring sperm DNA per every milliliter of Church buffer, and hybridized overnight at 65° C. with probes for HERV-K, HERV-W or L1 respectively.
  • HpaII methylation sensitive
  • Probe design was based on the hypothesis that relevant DNA methylation changes, if any, would include the predicted promoter regions of retrotransposons.
  • FIG. 2 shows the results obtained after using a probe for the promoter region of HERV-W.
  • FIG. 3 shows a Southern blot analysis of genomic DNA after digest with MspI (M) or its methylation-sensitive isoschizomer HpaIII (H), resp., hybridized with a LINE1 probe spanning the putative promoter region of the element. Equal amounts of DNA were loaded per sample, i.e. per MspI/HPaII pair. Fragment sizes range from 0.1 kb to >3.0 kb. Samples represent ovarian carcinoma (T—malignant), borderline ovarian tumor (B) and non-tumor ovarian tissue (N).
  • T malignant
  • B borderline ovarian tumor
  • N non-tumor ovarian tissue
  • Fragments between 1.4-2 kb as well as 0.4-0.7 kb (arrows) in HpaIII digests appear more pronounced in the malignant tissue samples compared to the non-tumor samples, indicating extensive cytosine methylation of this particular LINE1 region in non-carcinoma ovarian tissue and loss of LINE1 methylation in some ovarian carcinoma samples.
  • Southern Blot images are consistent with hypomethylation of Herv-W and LINE1 elements, respectively, in ovarian carcinoma versus normal ovarian tissue. The changes are more pronounced for Herv-W and more consistent among carcinoma samples. There is some heterogeneity for the effect among the samples tested, which will be correlated with clinical history of the tumors and treatment responses.
  • Retrotransposons have been identified as potential targets of hypomethylation during cellular transformation.
  • the following example provides the results of an examination of the methylation status of CpG dinucleotides associated with the L1 and HERV-W retrotransposons in benign and malignant human ovarian tumors.
  • a reduction in the methylation of CpG dinucleotides was found within the promoter regions of these retroelements in malignant relative to non-malignant ovarian tissues. Consistent with these results, it was also found that relative Li and human endogenous retrovirus-W (HERV-W) expression levels are elevated in representative samples of malignant vs. non-malignant ovarian tissues.
  • HERV-W human endogenous retrovirus-W
  • L1 is the most abundant family of human LINE elements comprising about 17% of the genome [22].
  • Human Endogenous Retrovirus-W (HERV-W) is a family LTR retrotransposons consisting of ⁇ 140 full-length or truncated elements randomly dispersed throughout the human genome [23]. These results demonstrate that large numbers of both families of retrotransposons are hypomethylated in ovarian carcinomas. It is further demonstrated that relative levels of both L1 and HERV-W expression are elevated in representative samples of malignant vs. non-malignant ovarian tissues. The findings presented herein are consistent with the hypothesis that retrotransposons are a major target of global hypomethylation associated with cellular transformation.
  • a restriction-enzyme based assay was utilized to compare the methylation status of CpG dinucleotides located within the promoter regions of these elements in a series of malignant and non-malignant ovarian tissues.
  • the restriction enzymes MspI and HpaII both recognize the sequence CCGG but HpaII only cuts when the recognition sequence is unmethylated at the inner cytosine (i.e., C C GG) while MspI is indifferent to the methylation status of the inner cytosine
  • FIGS. 4A & B displays Southern blots of HpaI and MspI digested genomic DNA isolated from tissue samples and hybridized against probes homologous to regions encompassing the promoter regions of each family of elements.
  • the HpaII/MspI restriction sites located within the promoter regions of both L1 and HERV-W elements are polymorphic among family members.
  • results presented in FIGS. 4A & B show that MspI-generated bands within the expected size range of internal fragments were visible in digestions of DNA from all tissue samples. In contrast, HpaII-generated fragments within the expected size range were only visible in digestions of DNA from the malignant samples. These results are indicative of a consistent reduction in the methylation of CpG dinucleotides within the promoter regions of both L1 and HERV-W elements in the malignant tissue. The fact that the number and intensity of HpaII generated bands in the malignant samples is significantly less than generated by MspI digestion indicates that some L1 and HERV-W elements remain hypermethlyated in the malignant samples. Regardless, this is the first report of the hypomethylation of L1 elements in ovarian carcinomas and of the hypomethylation of HERV-W in any human cancer.
  • hypomethylation is generally associated with the relaxation of chromatin structure, an increased accessibility of transcription factors and a consequent elevation in levels of expression [27]. These findings are generally consistent with these prior results. Since transcription is a rate limiting step in retrotransposition [11], hypomethylation might be expected to result in an increase in retrotransposon insertion mutations. While there have been occasional reports of L1 and other retrotransposon insertion mutations implicated in cancer development in humans [e.g., 28], this may not be as significant a factor as it apparently is in the mouse [29], perhaps because most L1 and other retrotransposon sequences in the human genome are believed to be truncated or otherwise transpositionally defective [30].
  • retrotransposons are known to be high affinity targets [e.g., 10].
  • Complexes released from retroelements may initiate a cascade of regulatory changes by binding to other lower affinity target sites and possibly resulting in the down regulation of genes essential for DNA repair and genome stability.
  • Genomic DNA was extracted by proteinase K digestion of 20-25 mg of bulk ovarian tissue and phenol-chlorophorm extraction. DNA was ethanol precipitated and re-suspended in water. Ten micrograms of genomic DNA were digested overnight at 37° C. with 10 to 16 excess amount of either HpaII [methylation sensitive restriction enzyme] or MspI [not sensitive for methylation at internal cytosine]. These enzymes recognize the sequence CCGG, which is found in diverse positions in the promoter regions of these retroelements. Digested DNA was resolved on an agarose gel and transferred to a nylon membrane (Hybond N; Amersham-Biosciences, Piscataway, N.J.) with NaOH.
  • Membranes were prehybridized for 1 hour with 10 mg/ml of herring sperm DNA in Church buffer [0.5M NaH 2 PO 4 , 7% SDS and 10M EDTA] and hybridized overnight at 65° C. in the same buffer with 100-200 ng of probe DNA labeled with [ ⁇ - 32 P]dCTP using a Nick Translation Kit (Roche, Indianapolis, Ind.). Filters were washed twice for 15 min in 2 ⁇ SSC and 0.1% SDS and then twice for 30 min in 1 ⁇ SSC and 0.1% SDS at 65° C. and exposed to Phosphorimager screens (Molecular Dynamics, Sunnyvale, Calif.).
  • the HERV-W probe was designed in the LTR region, downstream of the putative TTAAAT box. PCR was performed on genomic DNA with forward primer HERVF 5′-CCACCACTGCTGTTTGCCAC-3′ (SEQ ID NO: 771) and reverse primer HERVR 5′-GCCTCGTGTTCTCTGACCTGGGG-3′ (SEQ ID NO: 772), producing a 304 bp fragment.
  • the LINE1 probe for the promoter region was designed according to Takai et al [18].
  • PCR was performed on genomic DNA with forward primer L1F 5′-CGGGTGATTTCTGCATTTCC-3′ (SEQ ID NO: 773) and reverse primer L1R 5′-GACATTTAAGTCTGCAGAGG-3′ (SEQ ID NO: 774), giving a product of 540 bp.
  • PCR products were cloned into pCR2.1-TOPO and transformed into TOP10 E. coli cells (Invitrogen, Carlsbad, Calif.). Plasmids were extracted (Qiaprep Spin Miniprep Kit, Qiagen, Valencia, Calif.) and sequenced. Subsequent PCR reactions were performed on cloned plasmid DNA for both HERV-W and LINE1, and gel extracted PCR products were used as hybridization probes.
  • the HERV-W primers used were: forward; 5′-TTGGCGGTATCACAACCTCT-3′ (SEQ ID NO: 775) reverse; 5′-GTGACGATTCCGGATTGA-3′ (SEQ ID NO: 776); (product size:230 bp) based on the HERV-W sequence (GeneBank accession no. AC000064).
  • the LINE-1 primers were: forward 5′-TCATAAAGCAAGTCCTCAGTGACC-3′ (SEQ ID NO: 777); reverse 5′-GGGGTGGAGAGTTCTGTAGATGTC-3′ (SEQ ID NO: 778) (product size:165 bp) based on the LINE-1 sequence (GeneBank accession no. M80343).
  • Real-time monitoring of PCR reactions was performed using the DNA Engine Opticon 2 System (MJ Research, Waltham, Mass.) and the SYBR Green iQ dye (BioRad, Hercules, Calif.) [24].
  • Table 2 shows a ranking of relative retroelement expression values comparing benign (control) vs. malignant (cancer) samples obtaining via microarray analysis on a gene chip ( FIG. 5 ).
  • the results of this experiment show that some retroelement families show a significant increase in expression in cancer (Stage m ovarian carcinoma) vs. controls (negative values in Comparison Rank column), some show no net change (values in Comparison Rank column around 0) and some show a decrease in net levels (positive value in Comparison Rank column).
  • the changes in expression can be due to changes in chromatin structure.
  • this data set shows that there is a heterogeneous response in changes in chromatin structure in stage III tumors.
  • stage m tumor samples is not limited to a particular stage of type of cancer and is merely illustrative of the kind of changes in retroelement expression that can be analyzed by the methods of the present invention in order to diagnose, stage and treat any type of cancer.

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Cited By (5)

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WO2008134596A3 (fr) * 2007-04-25 2008-12-24 Wayne John Cancer Inst Utilisation d'adn line-i methylés et non methylés comme marqueur du cancer
EP2210954A1 (fr) * 2009-01-22 2010-07-28 Heinrich-Heine-Universität Düsseldorf Détermination du niveau de méthylation de l'ADN
WO2012048113A2 (fr) 2010-10-07 2012-04-12 The General Hospital Corporation Biomarqueurs de cancer
US20150065368A1 (en) * 2011-12-20 2015-03-05 Biomerieux Method for the in vitro diagnosis or prognosis of ovarian cancer
WO2020165361A1 (fr) * 2019-02-14 2020-08-20 Vib Vzw Biomarqueurs de rétrotransposon

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US8680065B2 (en) 2008-09-11 2014-03-25 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Oligonucleotides of human endogenous retrovirus 9 (ERV-9) long terminal repeat (LTR) and methods of use

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US20110159501A1 (en) * 2007-04-25 2011-06-30 Hoon Dave S B Use of methylated or unmethylated line-1 dna as a cancer marker
US20090068660A1 (en) * 2007-04-25 2009-03-12 John Wayne Cancer Institute Use of methylated or unmethylated line-1 dna as a cancer marker
WO2008134596A3 (fr) * 2007-04-25 2008-12-24 Wayne John Cancer Inst Utilisation d'adn line-i methylés et non methylés comme marqueur du cancer
US20110159502A1 (en) * 2007-04-25 2011-06-30 Hoon Dave S B Use of methylated or unmethylated line-1 dna as a cancer marker
US20110151465A1 (en) * 2007-04-25 2011-06-23 Hoon Dave S B Use of methylated or unmethylated line-1 dna as a cancer marker
US9850528B2 (en) 2009-01-22 2017-12-26 Heinrich-Heine-Universität Düsseldorf Determination of the normalized degree of DNA methylation
WO2010084154A3 (fr) * 2009-01-22 2010-12-02 Heinrich-Heine-Universität Düsseldorf Détermination du degré de méthylation de l'adn
CN102356160A (zh) * 2009-01-22 2012-02-15 杜塞尔多夫海因里希·海涅大学 Dna甲基化水平的测定
EP2210954A1 (fr) * 2009-01-22 2010-07-28 Heinrich-Heine-Universität Düsseldorf Détermination du niveau de méthylation de l'ADN
WO2012048113A2 (fr) 2010-10-07 2012-04-12 The General Hospital Corporation Biomarqueurs de cancer
WO2012048113A3 (fr) * 2010-10-07 2012-07-26 The General Hospital Corporation Biomarqueurs de cancer
CN103517990A (zh) * 2010-10-07 2014-01-15 通用医疗公司 癌症生物标志物
US11142800B2 (en) 2010-10-07 2021-10-12 The General Hospital Corporation Biomarkers of cancer
US20150065368A1 (en) * 2011-12-20 2015-03-05 Biomerieux Method for the in vitro diagnosis or prognosis of ovarian cancer
US11453920B2 (en) * 2011-12-20 2022-09-27 Biomerieux Method for the in vitro diagnosis or prognosis of ovarian cancer
WO2020165361A1 (fr) * 2019-02-14 2020-08-20 Vib Vzw Biomarqueurs de rétrotransposon

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